SUMMARY Mycobacterium tuberculosis (Mtb), the principal etiological agent of tuberculosis (TB), infects over one-third of humanity and is now the leading cause of infectious disease mortality by a single pathogen. Pyrazinamide (PZA) is a first-line sterilizing anti-tubercular drug that is anticipated to be an irreplaceable component of future TB treatment regimens. Previous studies have demonstrated that PZA is a pro-drug which is converted to the active form of pyrazinoic acid (POA) by the M. tuberculosis amidase PncA, and that loss of function mutations in pncA account for the vast majority of PZA resistance. Resistance to PZA in turn is associated with significantly higher treatment failures and relapse rates. Attempts to directly administer POA in humans and mice have been unsuccessful because POA cannot attain sufficient concentrations within Mtb at the infection site. Prodrug approaches employing simple POA esters have been unable to overcome the intrinsic drug disposition liabilities of POA as conventional prodrugs are rapidly hydrolyzed by serum esterases. In preliminary data, we have developed a novel prodrug strategy for POA through conjugation to a ?-lactam promoiety leading to selective release of POA by the unique mycobacterial beta-lactamase BlaC and not by serum esterases. This strategy overcomes PZA-resistance by circumventing PncA-mediated activation and renders blaC conditionally essential since loss of function mutations to blaC, restores susceptibility of Mtb to the ?-lactam promoiety. Consequently, we hypothesize the genetic barrier for development of spontaneous resistance to our POA-?-lactam conjugates will be high. The objectives of this application are to synthesize and evaluate ?-lactam prodrugs of POA, which are orally bioavailable and selectively release POA within Mtb- infected macrophages. We will accomplish the overall objectives of this application by pursuing two specific aims. In aim, we will synthesize ?-lactam prodrugs of POA, which are selectively activated by Mtb to release POA and not by commensal microbiota. The prodrugs will be biochemically and microbiologically characterized with a panel of ?-lactamases and microorganisms. In aim 2, we will measure prodrug stability in serum and stimulated gastric fluid and permeability in Caco-2 cells. We will next determine complete pharmacokinetic parameters and the extent of POA release by the gut microbiome. Finally, we plan to evaluate one compound in a murine TB infection model.